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No, this isn’t about the board game "Battleship." C4F6 is an antibody to a mutant form of superoxide dismutase 1 (SOD1). In people with a rare form of amyotrophic lateral sclerosis, every cell produces mutant SOD1, but only certain motor neurons succumb to the toxic protein. Researchers have been wondering why, and now C4F6 may help answer the question. Crucially, in both patients and animals that model ALS, the antibody recognizes only some motor neurons, despite the ubiquitous presence of mSOD1. Researchers in the laboratory of Jonathan Glass at Emory University in Atlanta, Georgia, report their findings in the March 19 Proceedings of the National Academy of Sciences online. Such antibodies to misfolded SOD1 might be useful as therapeutics, noted Daryl Bosco of the University of Massachusetts Medical Center in Worcester. Further, understanding C4F6’s targets may help identify people who would stand to benefit from this kind of treatment (see ARF related news story; ARF news story). Bosco was not involved in the study.

“The main import of this paper is that it showed that this antibody, which binds to mutant SOD1-G93A, also binds to other [SOD1] mutants, but the reactivity appears to be specific for spinal motor neurons,” said Neil Cashman of the University of British Columbia in Vancouver, Canada, who was not part of the research team. “Despite the fact that SOD1 is expressed ubiquitously…there is only an immunohistochemically recognized deposit in the cells that are vulnerable to ALS in the mouse model SOD1 ALS.”

"What is so special about those cells recognized by the antibody?" wondered study first author Terrell Brotherton. Solve that problem and you might understand why ALS specifically destroys motor neurons, she suggested. C4F6 is one of several antibodies designed against misfolded SOD1 (Rakhit et al., 2007; Liu et al., 2009; Gros-Louis et al., 2010; Forsberg et al., 2010; Kerman et al., 2010). C4F6 was raised to a specific SOD1 mutant, G93A (see Urushitani et al., 2007). To study C4F6, Brotherton analyzed how it binds to SOD1 in cultured cells, rodents, and people, and how this binding changed in the presence of hydrophobic versus hydrophilic conformations, and various SOD1 mutations.

She began with tissues from mice and rats overexpressing SOD1-G93A. C4F6 labeled skein-like inclusions in the mouse motor neurons, which eventually degenerate, but not sensory neurons, which do not. Only some motor neurons bound C4F6; one cell would take it up while its neighbors remained clear. C4F6 also bound skein-like inclusions in spinal cord samples from three people who had familial ALS caused by a different SOD1 mutation, A4V; the sample set did not include G93A patients. The antibody did not cross-react with inclusions in tissue from sporadic ALS patients or control subjects. This staining pattern resembles that seen with other SOD1 antibodies, Cashman said, depending on their epitopes. “There will be probes that will recognize mutant misfolded SOD1 and others that will recognize wild-type misfolded SOD1, and others that will recognize both,” Cashman predicted. The study authors hypothesize—though have no proof—that C4F6 recognizes a toxic form of SOD1 that appears specifically in the motor neurons that are on their way to cell death, but does not form in neurons that remain healthy. This cell-specific labeling was the most exciting result, Brotherton said, although she cannot explain how the C4F6 epitope might cause, or result from, neurodegeneration in cells vulnerable to SOD1 toxicity.

What could the C4F6 epitope be? Brotherton suspected it would be part of a misfolded peptide with two likely characteristics: unnaturally exposed hydrophobic regions, and a propensity to glom together into aggregates. The data surprised her on both counts. Using hydrophobic interaction chromatography, she found that C4F6 was more likely to interact with hydrophilic SOD1 molecules than hydrophobic ones. Solubility experiments, involving serially extracting proteins from spinal cords of late-stage SOD1-G93A animals in increasingly harsh solvents and then looking for protein recognized by C4F6 showed that it was the soluble form of SOD1 that bound the antibody. Although C4F6 was originally designed to recognize SOD1-G93A, it turned out to also label Chinese hamster ovary cells expressing SOD1-A4V, in keeping with the human results, as well as SOD1-G37R, but not SOD1-G93C or the wild-type protein. Thus, C4F6 appears to bind a soluble, hydrophilic mutant SOD1 conformation unique to certain spinal motor neurons.

Some three-dimensional conformation of mSOD1 in sickened motor neurons is likely part, but not the whole, of the C4F6 epitope. Defining the epitope “has been really tricky, and has frustrated a lot of people,” Brotherton said. She speculated that another factor, for example, a post-translational modification, might be necessary for C4F6 binding. For SOD1, these modifications include formation of a disulfide bridge and association with copper and zinc ions. Another possible explanation could be that C4F6 recognizes the epitope only when it is concentrated, such as in small, soluble oligomers, Cashman suggested. Bosco, based on her own work with C4F6 (see ARF related news story on Bosco et al., 2010), suggested that there is both a linear amino acid portion of the epitope, related to the G93A mutation, and a conformation-specific contribution. Unlike Brotherton, Bosco did see C4F6 binding wild-type SOD1 in some sporadic ALS samples. The reason for the discrepancy is unclear; perhaps it relates to the methods used, Bosco said.

Oxidation is one post-translational SOD1 modification that has been studied. C4F6 binds to oxidized, recombinant wild-type SOD1 in vitro, and researchers including Bosco report in the March 13 PNAS online that hyper-oxidized wild-type SOD1 shows up in sporadic ALS, specifically cases with bulbar onset. First author Stefania Guareschi and senior author Piera Pasinelli of Thomas Jefferson University in Philadelphia, Pennsylvania, studied lymphoblasts derived from people with sporadic ALS, and saw that in some of those, wild-type SOD1 had an abnormally high number of oxidized carbonyl groups. This over-oxidized SOD1 interacted with mitochondrial Bcl-2 to form a cytotoxic complex in human embryonic kidney cells, just as mutant SOD1 does (see ARF related news story on Pedrini et al., 2010). This was true of oxidized SOD1 from seven participants who had bulbar onset. Guareschi has since moved to the Italian Agency for Research on ALS in Milan.

A variety of SOD1 antibodies recognized the super-oxidized form, Pasinelli said, so she does not anticipate it would be among C4F6’s specific targets. Since C4F6 did not interact with wild-type SOD1 in Brotherton’s hands, researchers hoping to understand the enzyme’s role in sporadic ALS will likely need non-antibody tools, Pasinelli said. If her results bear out in a larger study, the over-oxidized SOD1 might serve as a biomarker for people with sporadic ALS who are likely to benefit from SOD1-targeted therapies, she speculated.

C4F6 itself is unlikely to identify the sought-after ALS biomarker (see ARF related news story). “I am not sure there would be enough SOD1 in the cerebrospinal fluid to detect,” said Brotherton, noting that transgenic mice express unnaturally high amounts. Cashman added that attempts to measure misfolded SOD1 in spinal fluid have had no success. “If it is there, it is at undetectable levels,” he said.

“These results indicate that C4F6 is exquisitely selective for at least a subset of the SOD1 pathology in ALS,” wrote Charles Glabe of the University of California, Irvine, in an e-mail to ARF. “Conformation-dependent antibodies have a lot to tell us about the mechanisms of pathogenesis in protein misfolding diseases,” he added. For example, Glabe’s A11 antibody has helped researchers identify different kinds of amyloid (see ARF related news story on Kayed et al., 2003; ARF related news story on Maezawa et al., 2008; ARF related news story on Ehrnhoefer et al., 2008). Brotherton’s work characterizing C4F6 is an important step to understanding SOD1 misfolding, Cashman said. “We are just discovering the rules of SOD1 misfolding…and we will need reagents to do that,” he said. Cashman predicts the enzyme’s contortions will turn out to be “hideously complex.”—Amber Dance

Comments on News and Primary Papers

This recent paper by Glass and coworkers reports further insight into the recognition of misfolded SOD1 by the conformation-dependent antibody C4F6. This antibody was originally isolated by Jean-Pierre Julien in 2007. This antibody was prepared by immunization of mice with recombinant human SOD1 containing the G93A mutation associated with familial ALS. It had weak immunoreactivity with wild-type SOD1, but it reacted with other mutant forms of SOD1 such as the G37R mutation, indicating that it recognizes a conformation-dependent epitope that does not depend on the specific amino acid substitution. Subsequent work showed that oxidation of wild-type SOD1 causes it to react with C4F6. Here the authors report that "C4F6 reacted only with mutant SOD1 and showed remarkable selectivity for disease-affected tissues and cells." Unaffected cells in adjacent regions were not immunoreactive, even though they expressed high levels of mutant SOD1. These results indicate that C4F6 is exquisitely selective for at least a subset of the SOD1 pathology in ALS.

Conformation-dependent antibodies are increasingly recognized as pathology-specific reagents that give researchers a clearer and more selective view of the pathological species of misfolded proteins and distinguish them from the natively folded protein. This is especially important in view of recent findings that the same protein can adopt several different conformations, analogous to prion stains, raising the question of whether the different strains may have different significance for pathogenesis. Conformation-dependent monoclonal antibodies can recognize and distinguish this structural variation. The immune system seems to be exquisitely sensitive to alterations in protein structure because of the strong immunological tolerance for natively folded proteins. The majority of antibodies produced against a pathologically misfolded protein immunogen appear to be conformation dependent, and this type of antibody has been raised against most of the proteins that are associated with protein misfolding diseases. How the epitopes that these antibodies recognize behave in experiments can be quite different. For example, routine sample preparation methods that denature proteins, such as SDS use and paraffin embedding or antigen retrieval, can either create or destroy immunoreactivity. The authors here show that SDS treatment of G93A SOD1 from unaffected tissue causes it to be recognized by C4F6, indicating that SDS induces the misfolding of mutant SOD1 to form the immunoreactive epitope. The opposite result can also be obtained, and protein denaturation can destroy the epitope and prevent immunodetection. This means that it is unwise to presume that normal laboratory procedures that denature proteins will be adequate to observe pathology-specific conformational epitopes. A good place to start is with tissue and protein in a native state and then optimize the methods for the specific detection of the pathologically misfolded species. Conformation-dependent antibodies have a lot to tell us about the mechanisms of pathogenesis in protein misfolding diseases if we only listen.